Flow cytometry-based cell sorting was first introduced to the research community more than 20 years ago. It is a technology that has been widely applied in many areas of life science research, serving as a critical tool for those working in fields such as genetics, immunology, molecular biology and environmental science. Unlike bulk cell separation techniques such as immuno-panning or magnetic column separation, flow cytometry-based cell sorting instruments measure, classify and then sort individual cells or particles serially at rates of several thousand cells per second or higher. This rapid “one-by-one” processing of single cells has made flow cytometry a unique and valuable tool for extracting highly pure sub-populations of cells from otherwise heterogeneous cell suspensions.
Cells targeted for sorting are usually labeled in some manner with a fluorescent material. The fluorescent probes bound to a cell emit fluorescent light as the cell passes through a tightly focused, high intensity, light beam (typically a laser beam). A computer records emission intensities for each cell. These data are then used to classify each cell for specific sorting operations. Flow cytometry-based cell sorting has been successfully applied to hundreds of cell types, cell constituents and microorganisms, as well as many types of inorganic particles of comparable size.
There are two basic types of cell sorters in wide use today. They are the “droplet cell sorter” and the “fluid switching cell sorter.” The droplet cell sorter utilizes micro-droplets as containers to transport selected cells to a collection vessel. The micro-droplets are formed by coupling ultrasonic energy to a jetting stream. Droplets containing cells selected for sorting are then electrostatically steered to the desired location. A droplet cell sorter can process selected cells at rates of tens of thousands of cells per second, limited primarily by the frequency of droplet generation and the time required for illumination.
The second type of flow cytometry-based cell sorter is the fluid switching cell sorter. Most fluid switching cell sorters utilize a piezoelectric device to drive a mechanical system which diverts a segment of the flowing sample stream into a collection vessel. Compared to droplet cell sorters, fluid switching cell sorters have a lower maximum cell sorting rate due to the cycle time of the mechanical system used to divert the sample stream. This cycle time, the time between initial sample diversion and when stable non-sorted flow is restored, is typically significantly greater than the period of a droplet generator on a droplet cell sorter. This longer cycle time limits fluid switching cell sorters to processing rates of several hundred cells per second. For the same reason, the stream segment switched by a fluid cell sorter is usually at least ten times the volume of a single micro-drop from a droplet generator. This results in a correspondingly lower concentration of cells in the fluid switching sorter's collection vessel as compared to a droplet sorter's collection vessel.
Flow cytometers are also applied widely for rapidly analyzing heterogeneous cell suspensions to identify constituent sub-populations. Fluorescently labeled monoclonal antibodies are often used as “markers” to identify immune cells such as T lymphocytes and B lymphocytes. For example, clinical laboratories routinely use this technology to count the number of “CD4 positive” T cells in HIV infected patients. They also use this technology to identify cells associated with a variety of leukemia and lymphoma cancers.
A detailed description of a prior art flow cytometry system is given in United States Published Patent Application No. US 2005/0112541 A1 to Durack et al.
Flow cytometers are often used to measure fluorescence emission intensity from single cells labeled with multiple fluorescent molecules. To obtain simultaneous labeling with the desired number of fluorescent molecules, it is often necessary to use multiple laser sources for excitation, with the excitation wavelength of each laser corresponding to that required to cause a particular fluorescent labeling molecule to fluoresce at a particular emission wavelength. In many cases, the emission spectra from fluorescent molecules excited by different lasers may overlap, causing confusion as to which fluorescent molecule is actually being detected. For example, the PE-Cy5 tandem conjugate is excited efficiently at λ=532 nm and emits at λ=675 nm, while APC is excited at λ=632 nm and also emits at λ=675 nm. When these two fluorescent dyes and lasers are used concurrently, it is necessary for the flow cytometer to employ some technique for accurately and independently quantifying the intensity of the two overlapping emissions.
This has been accomplished in the prior art by spatially separating the two focused laser beams so that each individual cell passes through the two excitation sources sequentially. Knowledge of the distance between the two excitation source focus points and the cell velocity allows the data acquisition system in the flow cytometer to correlate these temporally separated fluorescence measurements. To enable independent observation of the separate excitation laser spots, it is necessary to include a spatial filter in the emission collection optical path. This spatial filter limits the field of view of a photodetector so that most of the photons that strike it are from the emission produced by a single excitation laser spot. Typically, this spatial filter consists of a series of lenses that focus in and out of a pinhole.
The use of such spatial filtering presents many problems. Focusing through a pinhole attenuates the emission reducing the sensitivity of the system. The spatial separation between the two beams must be carefully adjusted and held constant with a high degree of accuracy. This significantly complicates optical alignment, especially in cases when more than two excitation sources are required. Very importantly, there will be times when two or more cells arrive with a close enough spacing that part or all of different cells are simultaneously in each of the two beams. Depending on the efficiency of the spatial filtering system, this can lead to varying degrees of inter-beam “crosstalk” in the detection system. Managing this “crosstalk” is a major aspect of the design of a spatial filtering system on a flow cytometers. Smaller pinholes produce greater spatial isolation, but reduce sensitivity since they attenuate the emission to a higher degree. Larger pinholes increase sensitivity, but also increase crosstalk among emission points. Furthermore, spatial separation of two beams requires that one or both of the beams be adjusted to a less than optimal position, off the axis of the fluorescence collection optic. This reduces the collection efficiency from that focus spot(s) and further compromises the sensitivity of the optical system to low level fluorescence emission. Because of this, spatial separation of the beams does not scale well with increased numbers of excitation lasers. It is not practical to use more than three separate excitation focus spots when using spatial filtering techniques. With three excitation focus spots, one can be located at the optimum focus point of the optic and the other two can be located above and below in the image plane of the optic. A fourth spot would fall out of the usable collection angle of most collection optics. Additionally, spatial filtering requires that two detectors be used to collect the emission for the same wavelength band. This introduces significant additional expense as the photodetector and all optical components in the optical train (lenses, optical filters, etc.), plus all detection path electronic components (amplifiers, analog-to-digital converters, etc.) must be duplicated.
There is therefore a need in the prior art for a system and method of collecting fluorescent emission intensity in a flow cytometer system having multiple excitation sources. The present invention is directed to meeting this need.